Cytochrome oxidase (COX), the terminal respiratory complex of mitochondria, is composed of 12-13 subunits three of which, encoded by mtDNA, form the catalytic core of the enzyme. In S. cerevisiae, biogenesis of the complex is governed by some 3 dozen nuclear genes. These code for the set of imported subunits and for various accessory factors involved in expression of the catalytic subunits, membrane insertion and maturation of subunit 2 (copper protein), heme A synthesis, copper homeostasis, and still other events that are poorly defined at present. During the past grant period we: 1) obtained evidence that Cox15p, ferredoxin and ferredoxin reductase constitute a three component monooxygenase that converts heme O to the immediate precursor of heme A, 2) identified two new genes (COX18, COX2O) responsible for promoting membrane insertion and maturation of subunit 2, 3) characterized suppressors of shy1 mutants whose phenotype implicate subunit 1 (heme protein) as the target of Shy1p action, 4) collaborated with Dr. Pierre Rustin and his colleagues in demonstrating that the COX deficiency of a family with a history of a fatal neuromyopathy is caused by a mutation in heme:famesyl transferase. Our goals for this coming period is to complete studies on the heme A biosynthetic pathway, to continue the analysis of shy1 mutants and the remaining dozen nuclear genes whose functions in COX assembly remain largely unknown. With respect to heme A biosynthesis, we intend to obtain more direct evidence for the participation of ferredoxin in hydroxylation of heme O, to purify and characterize Cox15p, and to identify the hydroxylated precursor of heme A. Secondly, we will strive to gain better insights into the function of Shy1p by examining the assembly intermediates in shy1 mutants through pulse-chase experiments and by searching for new suppressors. These studies should also shed light on the biochemical basis of Leigh's syndrome, which has been shown to be caused by mutations in SURF1, the human homologue of SHY1. Together with the ongoing functional analysis of other COX-specific genes, these studies are anticipated to fill the existent gaps in our understanding of how this important enzyme is assembled.